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A Chemotactic Signaling Surface on Chey Defined by Suppressors of Flagellar Switch Mutations S

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A Chemotactic Signaling Surface on Chey Defined by Suppressors of Flagellar Switch Mutations S JOURNAL OF BACTERIOLOGY, Oct. 1992, p. 6247-6255 Vol. 174, No. 19 0021-9193/92/196247-09$02.00/0 Copyright X 1992, American Society for Microbiology A Chemotactic Signaling Surface on CheY Defined by Suppressors of Flagellar Switch Mutations S. J. ROMAN, M. MEYERS,t K. VOLZ, AND P. MATSUMURA* Department ofMicrobiology and Immunology (MIC 790), University of Illinois, Box 6998, Chicago, Illinois 60680 Received 22 May 1992/Accepted 3 August 1992 CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression offliG mutations. Interestingly, these cheY mutations dominantly suppressed bothfiG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch. In Escherichia coli and Salmonella typhimurium, chemo- We have used genetic suppression, a useful tool for taxis is achieved by regulating the direction of flagellar investigation of protein interactions (for general reviews, see rotation (reviewed in reference 46). Flagellar rotation is references 7, 14, and 18; for specific examples, see refer- controlled by the flagellar motor switch apparatus, a multi- ences 1, 2, 12, 13, 19, 21, 23, 26, 29, 30, 32, 43, 49, and 50), component macromolecular structure composed of at least to study structure-function aspects of CheY activity at the three different protein subunits, FliG, FliM, and FliN (55, switch. A number of earlier suppression studies have dem- 56), which imparts either clockwise (CW) or counterclock- onstrated interactions between cheY and flagellar switch wise (CCW) rotation to the motor. CW flagellar rotation genes fliG, fliM, and fliN (37, 38, 44, 55). In this study, we results in a tumble in the swimming pattern, while CCW extended the genetics of the CheY-switch interaction to the rotation results in smooth swimming. By continually sam- mapping of dominant suppressors of flagellar switch muta- pling the environment over time and modulating flagellar tions on the molecular structure of CheY. The amino acid rotation accordingly, cells can move toward a more favor- residues shown here to be critical for CheY signaling cluster able, and away from a less favorable, environment. on one face of the CheY protein, suggesting that this region The chemotaxis (Che) proteins couple flagellar rotation to of CheY transduces chemotactic signals directly to the the environment by transducing chemotactic signals from switch. specific transmembrane chemoreceptors to the flagellar switch apparatus. One of these proteins, CheY, is a small (14-kDa), single-domain protein homologous to the regulator MATERIALS AND METHODS proteins of bacterial two-component sensory transduction systems (3) and is the only two-component signaling protein Reagents. Restriction enzymes and T4 DNA ligase were for which the high-resolution crystal structure has been purchased from Bethesda Research Laboratories (Gaithers- determined (47, 51). Like other regulators, CheY activity burg, Md.). Bacto-Tryptone, Bacto-Agar, and Bacto-Yeast appears to be controlled by phosphorylation (reviewed in Extract were from Difco Laboratories (Detroit, Mich.). Calf references 8 and 48); cheY function is required for CW intestinal alkaline phosphatase, used in some cloning exper- flagellar rotation (34, 35); CheY is phosphorylated in vitro by iments, was obtained from Boehringer Mannheim Biochem- CheA (16, 17, 33, 54); and cheY or cheA mutations that icals (Indianapolis, Ind.). Molecular biology grade agarose disrupt phosphotransfer reactions also result in smooth- was from International Biotechnologies, Inc. (New Haven, swimming phenotypes, suggesting that CheY-P is the active Conn.). Penicillin, hydroxylamine hydrochloride, protein CW generator (9, 33). While CheY is thought to act directly A-agarose, cyanogen bromide-activated Sepharose 4B, and on the switch apparatus to regulate flagellar rotation (11, 38, 3-3-indoleacrylic acid were obtained from Sigma Chemical 39, 53, 55), exactly how CheY functions is not known. Co. (St. Louis, Mo.). 125I-labeled protein A was purchased from ICN Biomedicals, Inc. (Costa Mesa, Calif.), and * Corresponding author. [y-32P]ATP was from Amersham Corp. (Arlington Heights, t Present address: Department of Biochemistry, Molecular Biol- Ill.). Other specialized enzymes and reagents are noted ogy and Cell Biology, Northwestern University, Evanston, IL where applicable. All other reagents and chemicals were of 60208. reagent grade and were obtained from Sigma Chemical Co., 6247 6248 ROMAN ET AL. J. BACT1ERIOL. TABLE 1. Bacterial strains and plasmids Strain or plasmid Characteristic(s) Source (reference); Strains RP437 Wild type (Che+) J. S. Parkinson RP4139 cheZ280 recA J. S. Parkinson RP4079 cheY216 recA J. S. Parkinson RP4650 cheB270 J. S. Parkinson RP5135 Atar-cheZ J. S. Parkinson RP4500 fliG1009 cheY+ J. S. Parkinson' RP4501 fliGlOlO cheY+ J. S. Parkinson' RP4503 fliGlO12 cheY+ J. S. Parkinson' RP4504 fliG1013 cheY+ J. S. Parkinson' RP4505 fliG1014 cheY+ J. S. Parkinson' RP4520 fliGlO29 cheY+ J. S. Parkinson' RP4496 fliMi005 cheY+ J. S. Parkinson RP4506 fliM1015 cheY+ J. S. Parkinson' RP4510 fliM1019 cheY+ J. S. Parkinson" RP4511 fliMi020 cheY+ J. S. Parkinson RP4517 fliM1026 cheYr J. S. Parkinson' AZ37 fliGlO37 cheYr This workb AZ38 fliGlO38 cheY+ This work" AZ39 fliG1O39 cheY+ This work' Plasmids pFZY oriF (low copy number) Penr A. Koop (20) via M. Winkler pMM1 tar operon (tar tap cheR cheB cheY cheZ flhB') in pFZY M. Meyers pRL22 Overproduces CheY and CheZ from tqp promoter R. Linzmeier pRL22AYC cheY deletion, overproduces CheZ only D. Vacante pRL22AZd cheZ deletion, overproduces CheY only This work pDV4 Overproduces CheA and CheW from trp promoter D. Vacante pYBO902 pMM1 cheY0902[T112I] This work pYBO903 pMM1 cheYO903[A9OV] This work pYBO904 pMM1 cheY0904[E117K] This work pYBO905 pMM1 cheYO905[E117K] This work pYBO906 pMM1 cheY0906[V108M] This work pYB1304 pMMl cheY1304[V11M] This work pYB1308 pMM1 cheY1308[T1121] This work pYB1409 pMM1 cheY1409[A9OT] Tlhis work pYB1610 pMM1 cheY1610[F111V] This work' pYB2885 pMM1 cheY2885[E27K] This worke pRYBO902d pRL22 cheY0902[T112I] This work pRYBO903d pRL22 cheYO903[A9OV] This work pRYBO904d pRL22 cheY0904[E117K] This work pRYBo9O5d pRL22 cheYO905[E117K] This work pRYBO906d pRL22 cheY0906[V108M] This work pRYB1304d pRL22 cheY1304[V11M] This work pRYB1308d pRL22 cheYl308[T112I] This work pRYB1409d pRL22 cheY1409[A90T] This work pRYB1610d pRL22 cheY1610[F111V] This work pRYB2885d pRL22 cheY2885[E27K] This work pRBB40.cheYD13K pRL22 derivative R. Bourret pRBB40.cheYD13KAZ AcheZ derivative This work pRBB40.cheYD57N pRL22 derivative R. Bourret pRBB40.cheYD57NAZ AcheZ derivative This work a J. S. Parkinson's original fliG (scyB) and fliM (scyA) allele numbers are retained as the last digits of our allele numbers. For example, fliGl100 = scyB1O, fliM1005 = scyAS, etc. b These strains were constructed in our laboratory from spontaneous chromosomal fliG suppressors of cheY304[Vl1M]. C pRL224Y, which was used to express CheZ, was produced by ExolIl-Sl digestion from the Sall site in cheY. In this construction, approximately 51 bp are deleted from cheY, resulting in an in-frame deletion that yields an internally deleted, nonfunctional CheY peptide (50a). d AZ versions of all high-copy-number cheY expression plasmids (both cheY+ and suppressor cheY alleles) were produced by PvuII deletion of cheZ. These plasmids were used to overproduce wild-type and suppressor CheY proteins in the absence of CheZ. The original strains with these cheY alleles are from J. S. Parkinson. The cheY alleles were cloned from the chromosome with mini-Mu and then subcloned into M13 and sequenced (5). To characterize dominant suppression, the cheY alleles were subcloned from mini-Mu into pMM1. Aldrich Chemical Co. (Milwaukee, Wis.), or Fisher Scien- tonA31 tsx-78), a Che+ reference strain that is itself a tific Co. (Fair Lawn, N.J.). derivative of E. coli K-12 (36). Switch mutant tester strains Bacterial strains and plasmids. Strains used in this study are Mot' Che- (motile but nonchemotactis), genotypically are listed with their relevant genotypes in Table 1. All strains denotedfliG cheY+ orfliM cheY+. They containfliG orfliM are derivatives of RP437 (F- thr-l(Amn) leuB6
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